What does it mean for purity to be "by area"?

That the percentage is calculated as the area of the main peak over the total area of the integrated peaks. It depends on the method and the detector; it is not the mass fraction of peptide in the vial.

Does a chromatogram with a single peak guarantee total purity?

No. An impurity can leave the column at the same time as the main compound, or not absorb at the detection wavelength, and go unseen.

Does the observed mass have to be identical to the theoretical one?

It must match within the instrument's tolerance. The COA should report both masses so that the reader can compare them.

Why do two COAs for the same compound show different purities?

Because the value depends on the method: column, gradient, detection and integration. Comparing them requires knowing both methods.

What is net peptide content?

The percentage of actual peptide within a weighed sample, not counting water or counterions. It is determined with methods such as amino acid analysis and is independent of HPLC purity.

Does a COA with HPLC and mass spectrometry certify sterility?

No. Sterility and endotoxins are determined with specific tests and can only be stated if the COA declares them.

HPLC and mass spectrometry in a peptide COA: what each one confirms

HPLC indicates what proportion of the detected signal corresponds to the main compound; mass spectrometry confirms that the measured mass matches the expected one. They complement each other: neither alone measures net peptide content, water, counterions or sterility. A useful COA reports the method and declares which tests were not performed.

What exactly does HPLC measure?

High-performance liquid chromatography (HPLC) separates the components of a sample by passing it through a column; peptides are usually analyzed in reverse phase with ultraviolet detection. The result is a chromatogram: a line with peaks, where each peak corresponds to a component that left the column at a given time.

The HPLC purity reported on a COA is usually an area percentage: the area of the main peak divided by the area of all integrated peaks. It is a relative measure of what the detector sees with the method used, not an absolute amount of peptide in the vial.

Chromatogram element	What it shows	What to ask
Retention time	The time at which each component leaves the column	Is it consistent between batches with the same method?
Main peak area	The signal of the main compound	What criterion was used for integration?
Secondary peaks	Impurities detectable with that method	Are they listed or only added together?
Baseline	The instrument noise	Is it clean, or are there unintegrated peaks?
Method	Column, mobile phase, gradient and detection	Is it declared in full so that it can be repeated?

Two laboratories can report different purities for the same batch because the value depends on the method: column, gradient, detection wavelength and integration criterion. A figure without a method cannot be compared with another.

What does mass spectrometry confirm and what does it not?

Mass spectrometry (MS) measures the mass-to-charge ratio of ionized molecules. For a peptide, the result is compared with the molecular mass expected for the declared sequence: if the observed mass matches within the instrument's tolerance, the identity is consistent with the declared compound.

A mass match is not the same as confirming the sequence. Two molecules with the same mass, for example isomers, cannot be told apart by mass alone. To confirm the sequence, peptide fragmentation (MS/MS) is used, an additional test that a standard COA does not always include.

That is why the COA should show the expected and the observed mass, and not just the word "confirmed".

Why are HPLC and mass spectrometry needed together?

Question	HPLC	Mass spectrometry
Is it the declared compound?	It does not confirm it on its own: two compounds can leave the column at the same time	Yes, as far as mass is concerned
What fraction is the main compound?	Yes, within what the detector sees	Not reliably on its own
Does it detect impurities with a different retention?	Yes	Only if coupled to chromatography (LC-MS)
Does it distinguish molecules of equal mass?	Sometimes, if they separate on the column	Not by mass alone

A COA with only HPLC reports the proportion of the main signal, but not which compound it is; one with only mass data reports which mass is present, but not in what proportion. Together they narrow down two separate questions: identity and chromatographic purity.

What do neither of them measure?

HPLC with ultraviolet detection and mass spectrometry do not cover several parameters that a laboratory may need. If the COA does not declare them, they are not backed up.

Parameter	How it is determined	Do HPLC or MS cover it?
Net peptide content	Amino acid analysis, among other methods	No
Water	Karl Fischer titration or another specific method	No
Counterions (for example trifluoroacetate or acetate)	Specific methods, often chromatographic	Only with a dedicated method, different from the purity one
Residual solvents	Gas chromatography or another specific method	No
Endotoxins	Bacterial endotoxins test	No
Sterility	Sterility test	No
Biological activity	Functional assay in the study system	No

Net peptide content is expressed as the percentage of actual peptide in a weighed sample and does not include the weight of water or counterions (Clinical Chemistry, 2016). That is why a lyophilized powder can have a total mass greater than the mass of peptide it contains.

What should the COA declare about its methods?

Batch number and analysis date.
For HPLC: column, mobile phase or gradient, detection and attached chromatogram.
For mass spectrometry: expected mass, observed mass and ionization technique.
Purity by area, with the integration criterion.
Parameters not determined, declared as such and not omitted.
Identifiable issuing laboratory.

The general fields of a COA and how to verify its authenticity are covered in this section's article on how to read a certificate of analysis.

Frequently asked questions

What does it mean for purity to be "by area"?: That the percentage is calculated as the area of the main peak over the total area of the integrated peaks. It depends on the method and the detector; it is not the mass fraction of peptide in the vial.
Does a chromatogram with a single peak guarantee total purity?: No. An impurity can leave the column at the same time as the main compound, or not absorb at the detection wavelength, and go unseen.
Does the observed mass have to be identical to the theoretical one?: It must match within the instrument's tolerance. The COA should report both masses so that the reader can compare them.
Why do two COAs for the same compound show different purities?: Because the value depends on the method: column, gradient, detection and integration. Comparing them requires knowing both methods.
What is net peptide content?: The percentage of actual peptide within a weighed sample, not counting water or counterions. It is determined with methods such as amino acid analysis and is independent of HPLC purity.
Does a COA with HPLC and mass spectrometry certify sterility?: No. Sterility and endotoxins are determined with specific tests and can only be stated if the COA declares them.

Sources

Related catalog references

Catalogued references from the families this document covers. Each sheet states presentations, current public price, and what batch documentation can be requested.

5-amino-1mqsmall molecule that inhibits the NNMT enzyme · Metabolic research peptides
CJC-1295 without DAC 5mg + Ipamorelin 5mgGHRH analog and secretagogue pentapeptide in the same vial · GH/IGF-1 axis and secretagogues
B7-33single-chain peptide derived from relaxin-2 · Tissue signaling and extracellular matrix
Adipotidepeptidomimetic with a prohibitin-binding domain · Metabolic research peptides
Follistatinactivin-binding glycoprotein · GH/IGF-1 axis and secretagogues

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Last reviewed: 2026-10-16. This site presents a technical catalog of references intended exclusively for research, technical or laboratory purposes. The published information is not medical advice, a prescription or a diagnosis, and the references are not for human or veterinary use. The availability, import, sale and dispatch of any reference are subject to document validation and to the health, customs and regulatory rules that apply in Colombia and in the destination country.